Mitochondrial metabolite compartmentalization in health and disease

项目概要 真核细胞在不同的细胞器中储存和利用代谢物——称为亚细胞代谢物 区隔化。不同的代谢酶和底物库提供了另一层灵活性 代谢物利用，从而能够对各种内在线索和外部压力进行强有力的适应。在 反过来，这些过程的缺陷与代谢紊乱有关，包括肥胖、胰岛素抵抗和 糖尿病。代谢物区室化的关键调节因子之一是线粒体转运蛋白：一个大的 许多载体蛋白，其中许多属于 SLC25A 蛋白家族，介导 代谢物穿过不可渗透的线粒体内膜并控制其在线粒体中的可用性 线粒体基质。然而，绝大多数线粒体 SLC25A 载体蛋白都是“孤儿” 转运蛋白，即它们的特定底物和生物功能仍然未知。 我们缺乏知识主要是由于许多线粒体膜蛋白不能被 在常规实验系统中正确重构，即使用重组蛋白制成的脂质体 在大肠杆菌或酵母中。为了解决这个问题，我们开发了一个强大的实验平台，使系统能够 使用棕色脂肪（最常见的线粒体转运蛋白之一）表征哺乳动物线粒体转运蛋白 富集细胞。我们将 CRISPRi 和 CRISPRa 系统整合到永生化棕色脂肪细胞中，这样 我们可以在小鼠和人类身上获得基本上无限量的“设计线粒体”。通过采用 新系统，我的实验室最近发现 SLC25A44 是哺乳动物中第一个线粒体 BCAA 转运蛋白， 这是该领域长期存在的一个谜（Yoneshiro et al. Nature 2019）。 该提案旨在生成线粒体 SLC25A 代谢物转运蛋白的完整功能图谱 哺乳动物。为了实现这一目标，我们计划将最先进的代谢组学和线粒体脂质体应用于 棕色脂肪衍生的设计师线粒体，并确定孤儿 SLC25A 载体的特定底物 蛋白质。我们将进一步确定孤儿SLC25A转运蛋白的生理和病理作用 体内，重点是代谢紊乱。该应用程序所产生的工作将建立一个概念性的 了解线粒体代谢物区室化的分子调控框架，以及 为逆转源于此类过程缺陷的疾病表型提供了新的路线图。

Project Summary Eukaryotic cells store and utilize metabolites in different organelles – referred to as subcellular metabolite compartmentalization. Distinct pools of metabolic enzymes and substrates provide another layer of flexibility in metabolite utilization, thereby allowing for robust adaptation to a variety of intrinsic cues and external stress. In turn, defects in the processes are associated with metabolic disorders, including obesity, insulin resistance, and diabetes. One of the critical regulators of metabolite compartmentalization is mitochondrial transporters: a large number of carrier proteins, many of which belong to the SLC25A protein family, mediate the translocation of metabolites across the impermeable mitochondrial inner-membrane and control their availability in the mitochondrial matrix. However, a vast majority of the mitochondrial SLC25A carrier proteins are “orphan” transporters, i.e., their specific substrates and biological functions remain unknown. The lack of our knowledge is primarily due to the fact that many mitochondrial membrane proteins cannot be reconstituted correctly in the conventional experimental system, i.e., liposomes using recombinant proteins made in E. Coli or yeast. To circumvent this issue, we developed a robust experimental platform that enables systemic characterization of mammalian mitochondrial transporters using brown fat, one of the most mitochondria- enriched cells. We incorporated the CRISPRi and CRISPRa system in immortalized brown adipocytes, such that we can obtain essentially unlimited amounts of “designer mitochondria” in mice and humans. By employing the new system, my lab has recently identified SLC25A44 as the first mitochondrial BCAA transporter in mammals, a long-standing mystery in the field (Yoneshiro et al. Nature 2019). This proposal aims to generate a complete functional map of mitochondrial SLC25A metabolite transporters in mammals. To achieve this goal, we plan to apply the state-of-art metabolomics and mitochondrial-liposomes to the brown fat-derived designer mitochondria, and to determine the specific substrates for orphan SLC25A carrier proteins. We will further determine the physiological and pathological roles of orphan SLC25A transporters in vivo, with an emphasis on metabolic disorders. The work resulting from this application will establish a conceptual framework to understand the molecular regulation of mitochondrial metabolite compartmentalization, and also provide a new roadmap for reversing disease phenotypes that stem from defects in such processes.